JPH09140394A - Production of optically active glycerol derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject compound useful as a raw material for a medicine, an agrochemical, a liquid crystal, etc., by treating a benzyloxy-3- chloro-2-propylsuccinic acid monoester with a bacterium or an enzyme having optically selectively hydrolyzing ability and removing the hydrolyzate. SOLUTION: A racemic modification of a 1-substituted or nonsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester of the formula (R is H or an alkyl) or an optically low-purity 1-substituted or nonsubstituted benzyloxy-3- chloro-2-propylsuccinic acid monoester of the formula is treated with an ester hydrolyzing enzyme derived from a bacterium belonging to the genus Serratia, Mucor, Rhizopus, Candida, Pseudomonas, Bacillus, Aspergillus or Staphylococcus or a bacterium containing the enzyme and a hydrolyzate is removed to give the objective compound of the formula useful as a physiologically active substance suitable as a medicine or an agrochemical or an intermediate for synthesizing a liquid crystal material, etc., efficiently and simply.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for obtaining a glycerol derivative having high optical purity by subjecting a racemic body or a glycerol derivative represented by the following formula (I) having low optical purity to microbial treatment or enzyme treatment.

[0002]

It is widely known that optical isomers exist in many biologically active compounds. And usually, the optical isomer exhibiting the desired biological activity is limited to one isomer, and the other optical isomer does not have the desired biological activity or is not preferable such as toxicity. It may have physiological effects.

In the case of a biologically active compound having such an optical isomer, if there is no suitable means or method for obtaining the desired optically active substance, the racemic compound is used as it is for the use of pharmaceuticals or agricultural chemicals. Is often used in. In such a case, there have been problems that the desired biological activity is significantly reduced and that an undesirable physiological action such as toxicity is accompanied.

In order to solve this problem, various methods for resolving optical isomers have been studied. For example, various ester derivatives of optical isomers to be resolved are prepared, an enzyme that optically and selectively hydrolyzes an ester is allowed to act on the ester derivative, and the difference in the solubility of the reaction system in a solvent is used to obtain an optical derivative. Methods have been proposed for separating isomers from each other. In order to realize this method, microorganisms producing ester degrading enzymes having various optical selectivity have been developed.

In such a technical background, an optically active glycerol derivative represented by the following formula (I) and an optically active glycidol derivative represented by the following formula (II) derived from, for example, alkali treatment are conventionally used. Therefore, it is important as an intermediate in the synthesis of useful bioactive substances for pharmaceuticals or agricultural chemicals or liquid crystal materials:

Embedded image

Embedded image [In the formula, R represents hydrogen or an alkyl group, and * represents the position of the asymmetric carbon atom].

It is known that these optically active glycerol derivatives and glycidol derivatives can be synthesized by using D-mannitol as a starting material [Journal of Organic Chemistry (J. Org. Chem.), 4
3, 4876 (1978)]. However, this method is not suitable for application to industrial production because the process is long and heavy metals must be used.

On the other hand, recently, production of an optically active glycerol derivative using an optically selective reaction of microorganisms and enzymes has been reported. For example, a method for producing an optically active 3-chloro-1,2-propanediol derivative utilizing microorganisms of the genus Corynebacterium, the genus Brevibacterium and the genus Micrococcus (JP-A-7-115991), trityl-1, by lipase, Optical selective esterification of 2-diol
[Journal of Organic Chemistry, 5
7, 1605 (1992)], optically selective esterification of 3-halo-2-acyloxy-1-alkylsulfonyloxypropane (JP-A-1-96176), 1-halogeno-3- (4-
Optically selective esterification of methoxyphenyloxy) -2-propanol (JP-A-5-194299), optically selective hydrolysis of 3-chloro-2-acetoxy-1-p-toluenesulfonyloxypropane (Japanese Patent Publication No. 4-7500).
0; Japanese Patent Publication No. 5-13636), optically selective hydrolysis of 2-butanoyl-1-phenylmethyl-3-chloro-1,2-propanediol [tetrahedron: asymmetry
(Tetrahedron: Asymmetry), 4, 961 (1993)] and the like are reported.

Further, production of an optically active glycidol derivative utilizing an optically selective reaction of microorganisms and enzymes has been reported. For example, optical selective hydrolysis of racemic glycidyl butyrate by porcine pancreatic lipase [Journal of American Chemical Society (J. Am. C.
hem. Soc.), 106, 7252 (1984); JP-A-1-28519.
9], Optical resolution of benzylglycidol precursor [Journal of Organic Chemistry (J. Org. Che
m.), 55, 4897 (1990)], or Acetobacter (A
Asymmetric oxidation by alcohol dehydrogenases such as genus cetobacter and genus Gluconobacter (EP
0 464 905 A1) has been reported.

However, these conventional methods utilizing microorganisms and enzymes can give glycerol derivatives and glycidol derivatives with high optical purity when compared with synthetic methods, but the enzymes used are expensive, This included problems such as the difficulty in mass production due to production technology.

[0010]

SUMMARY OF THE INVENTION The present invention has been made in view of the problems of the above-mentioned prior art, and the object thereof is represented by the above formula (I) having high optical purity. Another object of the present invention is to provide a method for producing a glycerol derivative and a glycidol derivative represented by the above formula (II) conveniently and efficiently industrially.

[0011]

Means for Solving the Problems As a result of various studies to solve the above problems, the present inventors have shown that the racemic compound or the 1-substituted or unsubstituted benzyloxy-group represented by the above (I) having a low optical purity is used. A specific ester-degrading enzyme or a microorganism containing the enzyme is allowed to act on 3-chloro-2-propylsuccinic acid monoester, and the hydrolysis product is removed to easily obtain the succinic acid monoester with high optical purity. I found that I could be.

That is, the present invention has the formula:

Embedded image [Wherein R represents hydrogen or an alkyl group] or a racemic compound represented by the formula 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester having low optical purity, and the succinic acid monoester Serratia, Mucoa, Rhizopus, Candida, Pseudomonas, Bacillus, which has the ability to optically selectively hydrolyze
An ester-degrading enzyme derived from a microorganism belonging to the genus Aspergillus or Staphylococcus, or a microorganism containing the enzyme is allowed to act to remove a hydrolysis product, which is represented by the formula (I) with high optical purity. The present invention provides a method for producing a 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester.

The glycidol derivative represented by the above formula (II) having a high optical purity is usually a 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester having a high optical purity obtained by this method. It can be manufactured by subjecting it to an alkali treatment.

In the present invention, R in formula (I) and formula (II)
Is an alkyl group, the alkyl group is 1 to 20
It may be a straight-chain or branched-chain alkyl group having 1, preferably 1 to 10 carbon atoms. More preferably, it may be a lower alkyl group having 1 to 4 carbon atoms, such as a methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl group. Further, this alkyl group may be bonded at any position of the ortho, meta and para positions.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The esterase used in the method of the present invention is an esterase having high optical selectivity to 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester. Is.
Such esterases are known as Serratia
Genus, Mucor genus, Rhizopus genus, Candida genus, Pseudomonas
Genus, genus Bacillus, genus Aspergi
llus) and Staphylococcus genus.

[0016] Preferably, Serratia marcescens
(Serratia marcescens), Mucor miehei
ehei), Rhizopus japonicu
s), Candida lipolytic
a), Pseudomonas fragi,
Bacillus subtilis, Aspergillus ochraceus, Bacillus licheniformi
s), Bacillus badius, Bacillus brevis, Bacillus circulans, Bacillus cereus (B)
acillus cereus), Bacillus megaterium (Bacillus
megaterium), and an esterolytic enzyme produced by a microorganism selected from the group consisting of Staphylococcus xylosus.

More preferably, Bacillus badius IAM 11059, Bacillus brevis IFO 33
31, Bacillus cereus IAM 1029, Bacillus circulans ATCC 9500, Bacillus circulans IFO 3329, Bacillus licheniformis IFO 12107, Bacillus licheniformis IFO 12195, Bacillus licheniformis I
FO 12199, Bacillus megaterium IAM 1
227, Bacillus subtilis IAM 1286, Bacillus subtilis IAM 1069, Bacillus subtilis IAM 1186, and Staphylococcus xylosus JCM 2418.

Further, the optically active 1 represented by the above formula (I)
-Substituted or unsubstituted benzyloxy-3-chloro-2-
In order to obtain an R-type monoester of propylsuccinic acid monoester, a microorganism belonging to the genus Bacillus, particularly Bacillus licheniformis IFO 12195, Bacillus licheniformis IFO 12199, Bacillus licheniformis IFO 12199, Bacillus circulans ATCC 9500 is used. , Bacillus circulans IFO 3329, Bacillus subtilis I
AM 1286, Bacillus subtilis IAM 10
69, and Bacillus subtilis IAM 1186
It is preferred to use an esterolytic enzyme produced by a microorganism selected from the group consisting of On the other hand, in order to obtain the S-form monoester, it is preferable to use a microorganism belonging to the genus Staphylococcus, particularly an ester-degrading enzyme produced by Staphylococcus xylosus JCM 2418.

These microorganisms can be obtained from the Institute for Applied Microbiology, University of Tokyo Microorganism Microalgae Center (IAM), Fermentation Research Institute (IFO), American Type Culture Collection (ATC)
C), or RIKEN Microorganism Preservation Facility (JCM)
Can be obtained from

In the method of the present invention, not only crude or purified esterases derived from the above microorganisms,
A microbial cell that produces the enzyme, a microbial cell culture, a treated microbial cell, or the like can also be used. In addition, microbial strains obtained by the artificial mutation method of these microorganisms can be used as long as they produce the enzyme.

Commercially available ester-degrading enzymes that can be used in the method of the present invention include SM esterase [derived from Serratia marcescens, manufactured by Nagase Seikagaku], Lipase F7 [derived from Mucoa miehei, Enzymatics ( Enzymatix)], Lilipase A5 [from Rhizopus japonicas, Nagase Seikagaku], Lipase F6 [from Candida lipolytica, manufactured by Enzymatics], Multifect P53 [from Bacillus subtilis, Gee]・ C-I (GCI)], Lipase B [Pseudomonas fragey-derived, Wako Pure Chemical Industries], biopsin [Aspazilas oclaseus-derived, Nagase Seikagaku], thermostable alkaline protease [derived from Bacillus genus, Kurita Water Industries Co., Ltd., and Talipase [Tanabe Pharmaceutical Co., Ltd.] Especially by using SM esterase or lipase F7, the above formula
Among the optically active 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoesters represented by (I), S-form monoesters can be efficiently obtained.

The above-mentioned microorganism is a commonly used solid medium,
Either liquid medium may be used for culturing, but liquid medium is preferable. The culture medium contains the usual carbon sources, nitrogen sources and minerals required for the growth and growth of microorganisms. As the carbon source, for example, starch, sucrose or glucose can be used. As the nitrogen source, natural nitrogen sources such as yeast extract, casein, corn steep liquor, peptone and meat extract, and inorganic nitrogen compounds such as ammonium sulfate, ammonium salt and urea can be used.
As the inorganic substance, an inorganic salt such as potassium phosphate, zinc sulfate or magnesium sulfate can be used.

The concentrations of carbon source, nitrogen source and inorganic substances in the medium are 1 to 20%, 1 to 20% and 1 to 100, respectively.
It may be in the range of 0 ppm, and the culture time is about 16 to 48 hours. Culture may be performed using any of static culture, aeration and agitation culture, or shaking culture.

Racemic 1-substituted or unsubstituted benzyloxy-3-, which is a substrate for the enzymatic reaction in the method of the present invention
Chloro-2-propylsuccinic acid monoester can be easily synthesized by a known method. For example, in toluene, racemic epichlorohydrin is reacted with a substituted or unsubstituted benzyl alcohol in the presence of BF ₃ .Et ₂ O to give 1-substituted or unsubstituted benzyloxy-3-chloro-2-propanol. To synthesize. Then, the product is esterified with succinic anhydride to obtain the desired succinic acid monoester.

In the present method, a racemic substrate is usually used. For example, the optical purity of one of the (S) -form and the (R) -form after the optical resolution treatment is low. It is also possible to use mixtures.

The method of the present invention is derived from the above microorganism in water or a buffer containing 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester having a racemate or low optical purity. It is carried out by adding an ester-degrading enzyme, a microorganism itself containing the enzyme or a culture thereof, and stirring. Reaction solution
The pH is 5-9, preferably 6-8. Reaction temperature is 5
C. to 50.degree. C., preferably 10.degree. C. to 30.degree. The amount of the esterolytic enzyme used in the reaction or the microorganism or its culture is not particularly limited, but the amount of the enzyme is 50 to 50 relative to the substrate 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester. It is preferable to add it in a range of 500 mg / mol of substrate. The reaction time varies depending on the amount of enzyme used, reaction temperature, reaction pH, etc., but is usually completed in about 1 to 24 hours.

After completion of the reaction, the 1-substituted or unsubstituted benzyloxy-3-chloro-2-propanol produced and the unreacted 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester were mixed with each other. Solvent extraction method,
It is separated by a commonly used separation operation such as a crystal precipitation method or a column chromatography method.

In the solvent extraction method, ethyl acetate was used under acidic conditions, and the 1-substituted or unsubstituted benzyloxy-3-chloro-2-propanol produced and unreacted 1-substituted or unsubstituted benzyloxy-3- Chloro-2-propylsuccinic acid monoester is extracted from the reaction solution. Then
By extracting only the unreacted 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester with a pH 8 potassium phosphate buffer solution, the recovery rate of this ester from the reaction solution was 95% or more. Can be collected at.

Further, an appropriate amount of alkali (eg, 2N sodium hydroxide) is added to an aqueous solution containing 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester to form an epoxy ring. By doing so, an optically active substituted or unsubstituted benzylglycidol can be produced in a yield of 95% or more.

These reactions are summarized in Reaction Formula I below.

Embedded image

[0031]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples. In addition, the quantification of 1-benzyloxy-3-chloro-2-propylsuccinic acid monoester and 1-benzyloxy-3-chloro-2-propanol in the reaction liquid and the measurement of the optical purity in the reaction solution in Examples were carried out by the following analysis. Conditions,
Performed by high performance liquid chromatography. High Performance Liquid Chromatography Analytical condition equipment: LC Module 1 High Performance Liquid Chromatography manufactured by Waters; Column: CHIRALPAK AS 0.46φ × 2
50 mm (manufactured by Daicel Chemical Industries, Ltd.); Eluent: hexane / isopropyl alcohol / acetic acid (9
7: 3: 0.3); temperature: room temperature; flow rate: 1 ml / min; detector: UV 254 nm.

Example 1 Culture of Microorganism The microorganism used in the method of the present invention was cultured as follows.
That is, in a 500 mL Erlenmeyer flask, a medium 1 having the following composition
After taking up 00 mL, 1 mL of a culture solution of the above-mentioned microorganism pre-cultured in the same medium was inoculated, and shake culture (160 rpm) was carried out at 30 ° C. for 16 hours on a rotary shaker. After completion of the culture, a cell culture solution having an OD ₆₆₀ of 10 to 30 was obtained.

[Table 1] Table 1. Medium composition Meat extract 5 g Peptone 15 g NaCl 15 g K ₂ HPO ₄ 5 g Olive oil 1 g Tween 80 1 g Water 1000 mL NaOH or HCl aqueous solution to adjust the pH to 7.0.

Example 2 (S) -1-benzyloxy-3
-Production of chloro-2-propyl succinic acid monoester
(No. 1) 1-Benzyloxy-3-chloro-2-propylsuccinic acid monoester was added to a vial containing 5 mL of 300 mM potassium phosphate buffer (pH 7.0) so as to have a concentration of 100 mM. To this, 50 mg of SM esterase derived from Serratia marcescens was added and stirred at 30 ° C. for 16 hours. After adding 1 mL of 2N hydrochloric acid to the reaction solution to stop the reaction, 1 mL of 1-benzyloxy-3-chloro-2-propanol and unreacted 1-benzyloxy-3-chloro-2-propylsuccinate were added with 5 mL of ethyl acetate. The acid monoester was extracted. Analysis of this extract by high performance liquid chromatography revealed that (S) -1-benzyloxy-3-chloro-2-propylsuccinic acid monoester having an optical purity of 99% was obtained in a yield of 45%. It was 50 mM potassium phosphate buffer (pH) was added to the ethyl acetate extract.
8.0) 5 mL was added, and (S) -1-benzyloxy-3-
Only chloro-2-propylsuccinic acid monoester was extracted to obtain (S) -1-benzyloxy-3- with an optical purity of 99%.
Yield of chloro-2-propylsuccinic acid monoester 41
%.

Example 3 (S) -1-benzyloxy-3
-Production of chloro-2-propyl succinic acid monoester
(Part 2) SM esterase 20 derived from Serratia marcescens
A 300 mM potassium phosphate buffer solution (pH 7.
0) To a vial containing 30 mL was added methyl isobutyl ketone (MIBK) in which 1-benzyloxy-3-chloro-2-propylsuccinic acid monoester was dissolved to a concentration of 2 M, and the mixture was added at 50 ° C. at 50 ° C. Stir for hours. The pH of the reaction solution was adjusted to pH 6.6 with 2N sodium hydroxide using a pH stat. After completion of the reaction, the pH was adjusted to 9 with 4N sodium hydroxide, and the MIBK layer containing 1-benzyloxy-3-chloro-2-propanol and unreacted (S)
The aqueous layer containing -1-benzyloxy-3-chloro-2-propylsuccinic acid monoester was separated. The aqueous layer was washed with an equal volume of MIBK and analyzed by high performance liquid chromatography to find that (S) -1-benzyloxy-3-chloro-2-propylsuccinic acid monoester with an optical purity of 99% was obtained in a yield of 43%. It turned out that it was obtained in.

Example 4 (R) -1-benzyloxy-3
-Preparation of chloro-2-propyl succinic acid monoester 1-benzyloxy-3-chloro-2-propyl was added to a vial containing 5 mL of 300 mM potassium phosphate buffer (pH 7.0) so as to have a concentration of 100 mM. Succinic acid monoester was added. Bacillus licheniformis IFO 12107 then cultured as described in Example 1.
1 mL of the culture solution of was added and stirred at 30 ° C. for 16 hours. After the reaction was stopped by adding 1 mL of 2N hydrochloric acid, 5 mL of ethyl acetate was added to 1-benzyloxy-3-chloro-2.
-Extract 1-benzyloxy-3-chloro-2-propylsuccinic acid monoester unreacted with propanol.
The extract was analyzed by high performance liquid chromatography to find that it had an optical purity of 98% (R) -1-benzyloxy-3.
-Chloro-2-propyl succinic acid monoester yield 3
It was found that it was obtained at 7%. To this ethyl acetate extract, 5 mL of 50 mM potassium phosphate buffer (pH 8.0) was added, and only (R) -1-benzyloxy-3-chloro-2-propylsuccinic acid monoester was extracted to give an optical purity of 99.
% (R) -1-benzyloxy-3-chloro-2-propylsuccinic acid monoester was obtained with a yield of 33%.

Example 5 Optically Active Benzyloxy-3-
Production of chloro-2-propylsuccinic acid monoester Various ester hydrolases shown in Table 2 below or various microorganisms shown in Table 3 below were reacted according to the method of Example 3 or 4 to give an optically active (S)- 1-benzyloxy-
3-chloro-2-propyl succinic acid monoester or
(R) -1-Benzyloxy-3-chloro-2-propylsuccinic acid monoester was obtained.

[Table 2]  Yield Optical purity S / R (%) (Ee%) SM esterase (Serratia marcescens) 45 99 S lipase F7 (mucoa miehei) 41 98 S lipase A5 (Rhizopus japonicas) 33 97 S lipase F6 (Candida lipolytica) 16 94 S talipase 16 92 S multifect P53 Bacillus subtilis) 12 90 S Lipase B (Pseudomonas frassie) 21 83 S Biopsin (Aspazilas ocraceus) 19 80 SHeat-resistant alkaline protease (genus Bacillus) 18 96 R ee% = enantiomeric excess (%)

[Table 3]  Yield Optical purity S / R (%) (Ee%) Bacillus badius IAM 11059 40 88 88 R Bacillus brevis IFO 3331 36 90 R Bacillus cereus IAM 1029 36 92 R Bacillus circulans ATCC 9500 34 98 R Bacillus circulans IFO 3329 34 94 R Bacillus lichenifor 12 Bacillus licheniformis IFO 12195 36 96 R Bacillus licheniformis IFO 12199 33 96 96 R Bacillus megaterium IAM 1227 35 94 R Bacillus subtilis IAM 1286 34 98 R Bacillus subtilis IAM 1069 32 18 6 subtilis 6 R RStaphylococcus xylosus JCM 2418 18 95 S

Production Example 1 Production of (S) -benzylglycidol The (S) -1-benzyloxy-3-chloro-2-propylsuccinic acid monoester having an optical purity of 99% obtained in Example 3 was added at 5 ° C. Below, 5 equivalents of 2N sodium hydroxide was added and reacted for 5 hours. After the reaction was completed, the produced (S) -benzylglycidol was extracted with methyl-t-butyl ether and concentrated to obtain (S) -benzylglycidol with an optical purity of 98% ee or higher in a yield of 94%. It was

Production Example 2 Production of (R) -benzylglycidol The (R) -1-benzyloxy-3-chloro-2-propylsuccinic acid monoester having an optical purity of 99% obtained in Example 4 was added at 5 ° C. Below, 5 equivalents of 2N sodium hydroxide was added and reacted for 5 hours. After completion of the reaction, the produced (R) -benzylglycidol was extracted with methyl-t-butyl ether and concentrated to obtain (R) -benzylglycidol with an optical purity of 98% ee or higher in a yield of 92%. It was

[0039]

INDUSTRIAL APPLICABILITY As described above, the use of the method of the present invention makes it possible to easily and efficiently produce a glycerol derivative having a high optical purity, and this method is industrially extremely advantageous. In addition, a corresponding glycidol derivative having high optical purity can be easily produced by using the obtained glycerol derivative having high optical purity.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location (C12P 41/00 C12R 1: 085) (C12P 41/00 C12R 1:09) (C12P 41/00 C12R 1:10) (C12P 41/00 C12R 1:11) (C12P 41/00 C12R 1: 125) (C12P 41/00 C12R 1:44) (C12P 41/00 C12R 1:43) (C12P 41/00 C12R 1: 785) (C12P 41/00 C12R 1: 845) (C12P 41/00 C12R 1:73) (C12P 41/00 C12R 1:38) (C12P 41/00 C12R 1:66) (72) Inventor Shunji Kamiyama 2-3-2 Muroya, Nishi-ku, Kobe-shi, Hyogo Nagase & Co., Ltd. R & D Center

Claims (4)

[Claims] 1. The formula: embedded image [Wherein R represents hydrogen or an alkyl group] or a racemic compound represented by the formula 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester having low optical purity, and the succinic acid monoester Serratia, Mucoa, Rhizopus, Candida, Pseudomonas, Bacillus, which has the ability to optically selectively hydrolyze
An ester-degrading enzyme derived from a microorganism belonging to the genus Aspergillus or Staphylococcus or a microorganism containing the enzyme is allowed to act to remove a hydrolysis product, which is represented by the formula (I) with high optical purity. Process for producing 1-substituted or unsubstituted benzyloxy-3-chloro-2-propylsuccinic acid monoester. 2. The microorganisms are Serratia marcescens, Mucoa miehei, Rhizopus japonicas, Candida lipolytica, Pseudomonas frazi, Bacillus subtilis, Aspasillus ocraceus, Bacillus licheniformis, Bacillus badius, Bacillus brevis, The method according to claim 1, wherein the microorganism is selected from the group consisting of Bacillus circulans, Bacillus cereus, Bacillus megaterium, and Staphylococcus xylosus. 3. The microorganism is Bacillus badius IA.
M 11059, Bacillus brevis IFO 333
1. Bacillus cereus IAM 1029, Bacillus
Circulence ATCC 9500, Bacillus circulance IFO3329, Bacillus licheniformis IFO 12107, Bacillus licheniformis
IFO 12195, Bacillus licheniformis IF
O 12199, Bacillus megaterium IAM 12
27. The method according to claim 2, which is No. 27, Bacillus subtilis IAM 1286, Bacillus subtilis IAM 1069, Bacillus subtilis IAM 1186, or Staphylococcus xylosus JCM 2418. 4. The process according to claim 1, wherein the esterase is SM esterase, lipase F7, lipase A5, lipase F6, multifect P53, lipase B, biopsin, thermostable alkaline protease, or talipase.